Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria. S J Brand, D Stone J Clin Invest. 1988;82(3):1059-1066. https://doi.org/10.1172/JCI113662. Research Article Gastric acid exerts a feedback inhibition on the secretion of gastrin from antral G cells. This study examines whether gastrin gene expression is also regulated by changes in gastric pH. Achlorhydria was induced in rats by the gastric H+/K+ ATPase inhibitor, omeprazole (100 mumol/kg). This resulted in fourfold increases in both serum gastrin (within 2 h) and gastrin mRNA levels (after 24 h). Antral somatostatin D cells probably act as chemoreceptors for gastric acid to mediate a paracrine inhibition on gastrin secretion from adjacent G cells. Omeprazole-induced achlorhydria reduced D-cell activity as shown by a threefold decrease in antral somatostatin mRNA levels that began after 24 h. Exogenous administration of the somatostatin analogue SMS 201-995 (10 micrograms/kg) prevented both the hypergastrinemia and the increase in gastrin mRNA levels caused by omeprazole-induced achlorhydria. Exogenous somatostatin, however, did not influence the decrease in antral somatostatin mRNA levels seen with achlorhydria. These data, therefore, support the hypothesis that antral D cells act as chemoreceptors for changes in gastric pH, and modulates somatostatin secretion and synthesis to mediate a paracrine inhibition on gastrin gene expression in adjacent G cells. Find the latest version: https://jci.me/113662/pdf Reciprocal Regulation of Antral Gastrin and Somatostatin Gene Expression by Omeprazole-induced Achlorhydria Stephen J. Brand and Deborah Stone Departments ofMedicine, Harvard Medical School and Massachusetts General Hospital, Gastrointestinal Unit, Boston, Massachusetts Abstract Substantial evidence supports the hypothesis that gastric acid inhibits gastrin secretion through somatostatin released Gastric acid exerts a feedback inhibition on the secretion of from antral D cells (9, 10). Somatostatin is a potent inhibitor gastrin from antral G cells. This study examines whether gas- of gastrin release (11). Antral D cells have characteristic cyto- trin gene expression is also regulated by changes in gastric plasmic projections onto G cells (12, 13) that may act as a pH. Achlorhydria was induced in rats by the gastric H+/K+ pathway for paracrine inhibition by somatostatin. Since gastric ATPase inhibitor, omeprazole (100 gmol/kg). This resulted in acid stimulates somatostatin release (14-16), gastrin secretion fourfold increases in both serum gastrin (within 2 h) and gas- is thought to be inhibited by somatostatin released locally from trin mRNA levels (after 24 h). these cytoplasmic processes. The inhibition of gastrin secretion Antral somatostatin D cells probably act as chemorecep- by antral somatostatin thus represents a probable example of tors for gastric acid to mediate a paracrine inhibition on paracrine regulation. Measurement of changes in somatostatin gastrin secretion from adjacent G cells. Omeprazole-induced mRNA levels with changes in gastric pH may be of value in achlorhydria reduced D-cell activity as shown by a threefold evaluating the D cell's role as a paracrine regulator of antral G decrease in antral somatostatin mRNA levels that began after cells, since these mRNA levels directly reflect D cell activity. 24 h. Exogenous administration of the somatostatin analogue Although somatostatin's inhibitory action on gastrin se- SMS 201-995 (10 gg/kg) prevented both the hypergastrin- cretion is well established, it is not known whether somato- emia and the increase in gastrin mRNA levels caused by ome- statin also inhibits gastrin gene expression. In the pituitary, prazole-induced achlorhydria. Exogenous somatostatin, how- where somatostatin also mediates a similar feedback cycle in- ever, did not influence the decrease in antral somatostatin volving the inhibition of hormone secretion (17), short term mRNA levels seen with achlorhydria. incubation with somatostatin does not inhibit growth hor- These data, therefore, support the hypothesis that antral D mone gene transcription (18). This suggests a dissociation be- cells act as chemoreceptors for changes in gastric pH, and tween somatostatin's ability to inhibit pituitary hormone se- modulates somatostatin secretion and synthesis to mediate a cretion and its effects on hormone synthesis. Consequently, paracrine inhibition on gastrin gene expression in adjacent somatostatin may not necessarily inhibit gastrin gene expres- G cells. sion despite its well described effects on gastrin secretion. Introduction Secretion of gastrin is inhibited by gastric acid, and this serves Methods as a negative feed back control that prevents excess acid secre- tion (1). Achlorhydria, as is seen in atrophic gastritis, releases Omeprazole treatment. Male Sprague-Dawley rats weighing 180-220 g antral gastrin cells (G cells) from this inhibition and conse- were fed regular laboratory chow ad lib. Omeprazole dissolved in 100 ,gmol/kg PEG 2000 (concentration 20 mg/ml) was injected intraperito- quently causes marked hypergastrinemia and G-cell hyperpla- neally twice daily at 12-h intervals (0.2 ml PEG in 1 ml 0.15% sia (1-3). Surgical ablation of gastric acid secretion in rats by NaHCO3). Control rats received intraperitoneal injections of 0.2 mls fundectomy produces similar changes (4) and has also been PEG alone in 1 ml NaHCO3. After an overnight fast, rats were anesthe- shown to stimulate gastrin synthesis (5). Chronic blockade of tized with ether, 1.5 ml of their blood was drawn by cardiac puncture, acid secretion with the H+/K+ ATPase antagonist omeprozole and then they were killed. The antrum was excised, and a similar results in hypergastrinemia and, with prolonged treatment, in weight of corpus from the greater curvature adjacent to the saccus- G-cell hyperplasia (6-8). These results suggest that achlorhy- corpus margin was excised. The duodenum, excluding a 2-mm rim dria may also stimulate gastrin gene expression in G cells. adjacent to the pylorus, was also removed. Tissues were immediately Rapidly inducing achlorhydria with omeprazole allows this placed in guanidium thiocyanate lysis buffer (18) and homogenized question to be addressed without using a polytron (Brinkmann Instruments Co., Westbury, NY). the complication of G cell RNA preparation. RNA was prepared by the lithium chloride pre- hyperplasia and should, therefore, illuminate the physiological cipitation method of Cathala et al. (19) and the concentration of RNA mechanisms that regulate gastrin gene expression. was measured spectrophotometrically by the absorbance at 260 A. The efficiency of RNA recovery, determined by adding 3H-labeled SP6 sense gastrin cRNA, was between 80-85%. Received for publication 13 July 1987 and in revised form I March RNA probe synthesis. 32P-labeled RNA probes were synthesized 1988. using SP6 RNA polymerase (20) transcribed from rat gastrin (21) and somatostatin (22) cDNA templates. Synthetic oligonucleotides com- 1. Abbreviations used in this paper: POMW, proopiomelanocortin. plementary to rat actin mRNA (TGCTGCAGTCTAGAGGATCC- GAATTCG) and 18S subunit ribosomal RNA were end labeled with J. Clin. Invest. 32p y ATP using polynucleotide kinase (108 cpm/lg sp act). © The American Society for Clinical Investigation, Inc. Quantitation ofgastrin and somatostatin mRNA by dot-blot analy- 0021-9738/88/09/1059/08 $2.00 sis. Total RNA was immobilized on to nitrocellulose filters (Schleicher Volume 82, September 1988, 1059-1066 & Schuell, Inc., Keene, NH) using a dot-blot procedure (23). As an Regulation of Gastrin Gene Expression by Gastric Achlorhydria 1059 internal correction for RNA loading variations, dot-blot filters were mRNA determination. Treatment with omeprazole resulted in hybridized with a 32P-labeled synthetic oligonucleotide that is comple- a marked hypergastrinemia (Fig. 1 A), serum gastrin being mentary to 18S ribosomal RNA. The baked filters were prehybridized fivefold higher in omeprazole-treated rats. Omeprazole treat- at 430C for several hours in sealed plastic bags containing 5 ml of 5X ment also increased gastrin mRNA levels as shown by North- standard saline citrate 0.2% 0.2% (SSC), Ficoll, polyvinyl pyrroline, ern 0.2% BSA, 50 mM sodium phosphate buffer, pH 7.0, 1% SDS, and blot analysis (Fig. 1 C) and dot-blot quantitation (Fig. 2 B). 0.1% sodium pyrophosphate-sonicated salmon sperm DNA (100 Northern blot analysis of antral RNA (Fig. 1 C) shows a single ug/ml). The 32P-labeled probe was added (106 cpm/ml) and the mix- gastrin mRNA species (- 570 nucleotides). The intensity of ture was hybridized at 550C for 15 h. Filters were then washed in 0.2X gastrin mRNA hybridization was increased in RNA from SSC, 1% SDS at 550C three times for 20 min each time. Filters were omeprazole-treated rats compared with control. Densitomet- exposed to x-ray film (Kodak AR- IO) for 6 and 18 h. Exposed film was ric quantitation showed that omeprazole induced an increase developed and fluorograms were quantitated by scanning with a laser in gastrin mRNA levels. Gastrin mRNA levels were 0.1 1±0.05 densitometer (LKB Instruments, Inc., Gaithersburg, MD) using an O.D. units for control RNA and 0.66±0.07 O.D. units for area integrator. The ribosomal RNA probe was then melted off by omeprazole-treated RNA, mean±SE, n = 10, P < 0.01. To heating blots at 950C in 0.1 X SSC for 15 min. The blots were then determine whether the increase in gastrin mRNA induced by exposed overnight to confirm the melting of the probe. Dot blots were then rehybridized with 32P-labeled RNA SP6 probes omeprazole was specific, actin mRNA levels were measured that were complementary to rat gastrin or somatostatin mRNA. Prehy- on the same blotted RNA samples (Fig. 1 B). In contrast to bridization and hybridization were performed as above with the fol- gastrin mRNA, actin mRNA levels were not significantly in- lowing exceptions: hybridization solution contained 50% formamide, creased in RNA from omeprazole-treated rats. Actin mRNA and 0.1% SDS, prehybridization temperature was 450C, and hybrid- levels were 0.29±0.03 O.D.